The present invention is directed to mutations of arginine deiminase for more efficient production and processing and thus improved treatment of cancer and other disease states.
It is presently believed that the amino acid arginine may play an important role in mediating certain disease states. For example, it has been reported that tumors such as melanomas, hepatomas, sarcomas and leukemia require arginine for growth. Sugimura et al., Melanoma Res. 2:191-196(1992); Takaku et al., Int. J. Cancer 51:244-249(1992); Miyazaki et al, Cancer Res. 50:4522-4527 (1990); J. B. Jones, xe2x80x9cThe Effect of Arginine Deiminase on Murine Leukemic Lymphoblasts,xe2x80x9d Ph.D. Dissertation, The University of Oklahoma, pages 1-165 (1981). Malignant melanoma (stage 3) and hepatoma are fatal diseases which kill most patients within one year of diagnosis. In the United States, approximately 16,000 people die from these diseases annually. The incidence of melanoma is rapidly increasing in the United States and is even higher in other countries, such as Australia. The incidence of hepatoma, in parts of the world where hepatitis is endemic, is even greater. For example, hepatoma is one of the leading forms of cancer in Japan and Taiwan.
It has also been reported that many protozoa require arginine for growth and thus arginine may play an important role in many parasitic diseases. van Wagtendonk et al., xe2x80x9cNitrogen Metabolism in Protozoaxe2x80x9d, in Comparative Biochemistry of Nitrogen Metabolism, pages 1-56 (J. W. Campbell ed. 1970). Arginine (derived from the circulation) has also been shown to be a source of nitrous oxide which can play an important role in mediating septic shock. Chang et al., Am J. Physiol. 274:H342-H348 (1998); McDonald et al., J. BioL Chem. 272:31213-31216 (1997). Effective treatments for these diseases are urgently needed.
It has been reported that enzymes which degrade non-essential amino acids, such as arginine, may be an effective means of controlling some forms of cancer. For example, arginine deiminase isolated from Pseudomonas putide was described by J. B. Jones, xe2x80x9cThe Effect of Arginine Deiminase on Murine Leukemic Lymphoblasts,xe2x80x9d Ph.D. Dissertation, The University of Oklahoma, pages 1-165 (1981). Because arginine deiminase catalyzes the conversion of arginine into citrulline, thus helping to eliminate arginine from the circulation of animals, it is believed that arginine deiminase can be used as an effective therapy for cancer and other disease states where arginine plays a role. Although arginine deiminase is not produced in mammals, it is found in a variety of bacteria, fungi and mycoplasma. Arginine deiminase can be thus be isolated from those organisms which produce it or, in the alternative, the enzyme may be produced using recombinant DNA technology. Misawa et al., J. Biotechnol. 36:145-155 (1994).
Certain disadvantages have come to be associated with the isolation of arginine deiminase from organisms. Although effective in killing tumor cells in vitro, arginine deiminase isolated from Pseudomonas pudita failed to exhibit efficacy in vivo because it had little enzyme activity at a neutral pH and was rapidly cleared from the circulation of experimental animals. Arginine deiminase derived from Mycoplasma arginini (SEQ ID NO:5) is described, for example, by Takaku et al, Int. J. Cancer, 51:244-249 (1992), and U.S. Pat. No. 5,474,928, the disclosures of which are hereby incorporated by reference herein in their entirety. A problem associated with the therapeutic use of such a heterologous protein is its antigenicity. The chemical modification of arginine deiminase from Mycoplasma arginini, via a cyanuric chloride linking group, with polyethylene glycol was described by Takaku et al., Int. J. Cancer Res. 84:1195-1200 (1993). The modified protein was toxic when metabolized due to the release of cyanide from the cyanuric chloride linking group.
The production of arginine deiminase via recombinant DNA techniques also provides for certain disadvantages. For example, arginine deiminase produced in Escherichia coli is enzymatically inactive and thus must be denatured and then properly renatured in order for it to become enzymatically active. The usual method for renaturing arginine deiminase produced in E. coli is to isolate the inactive enzyme, dissolve it in guanidinium hydrochloride and renature it by rapid dilution into low ionic strength buffer. This last step requires very large volumes of buffer thus making the manufacture of arginine deiminase both expensive and time consuming. However, recombinant technology does have certain advantages. For example, organisms more amenable to fermentation can be used as hosts. Additionally, these fermentation hosts are generally much less pathogenic and larger amounts of arginine deiminase can be obtained. It has been shown the E. coli may produce large amounts of Mycoplasma arginine deiminase.
Another problem associated with arginine deiminase is that the enzyme is highly antigenic and thus rapidly cleared from circulation. Accordingly, arginine deiminase must be properly formulated before being used as a therapeutic agent. For purposes of the present invention, the term formulation can be defined as the chemical modification of any arginine deiminase for purposes of reducing antigenicity of the enzyme. For example, it has been shown that the formulation of several proteins including arginine deiminase with polyethylene glycol, i.e. pegylation, can reduce the antigenicity of the protein and greatly increase its circulating half-life. Unfortunately, the formulation of arginine deiminase with polyethylene glycol often inactivates the enzyme.
There is a need for methods and compounds which address these problems associated with the prior art. The present invention is directed to these, as well as other, important ends.
The present invention relates to a modified arginine deiminase enzyme capable of more efficient production and processing.
The present invention further relates to a recombinant DNA molecule which encodes modified arginine deiminase.
In related aspects, the present invention further relates to recombinant vectors which comprise a nucleotide sequence that encodes modified arginine deiminase and to host cells comprising such vectors.
The present invention further relates to methods for preparing the modified arginine deiminase enzyme of the invention. These methods comprise growing a host cell transformed with the recombinant DNA molecule of this invention in a suitable culture medium.
In other aspects, this invention to methods of treating cancer as well as treating and/or inhibiting the metastasis of tumor cells. The invention also relates to methods of treating parasitic disease, septic shock and other disease states.
These and other aspects of the present invention will be elucidated in the following detailed description of the invention.